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PubMed1974

Differential thermolability of exonuclease and endonuclease activities of the recBC nuclease isolated from thermosensitive recB and recC mutants.

The recBC nuclease (also called exonuclease V) has been partially purified from Escherichia coli K-12 strains carrying the thermosensitive recB270, recC271, and recB270 recC271 mutations. Of the multiple activities associated with the enzyme, only the adenosine 5'-triphosphate-dependent exonucleolytic hydrolysis of duplex deoxyribonucleic acid (DNA) is abnormally thermolabile. The exo- and endonucleolytic degradation of single-stranded DNA is no more thermosensitive than that catalyzed by the wild-type enzyme. These results suggest that the defects in genetic recombination, DNA repair, and the maintenance of cell viability observed in recBC mutants in vivo result primarily from the specific loss of adenosine 5'-triphosphate-dependent exonuclease active on duplex DNA.

Adenosine Triphosphate↗

PubMed1996

Use of NASBA RNA amplification for detection of Mycobacterium leprae in skin biopsies from untreated and treated leprosy patients.

This study was performed to assess the value of NASBA RNA amplification of a 16S rRNA target for the detection of presumably viable Mycobacterium leprae in sections of skin biopsies from leprosy patients. The NASBA positivity rate was 90.4% (84/93) for untreated multibacillary (MB) patients [bacterial index (BI) > or = 2] and 16.7% (8/48) for the untreated paucibacillary (PB) patients (BI < 2). NASBA positivity showed a good concordance with the presence of solidly stained M. leprae [morphological index (MI)] in skin biopsies from leprosy patients, but no relationship could be demonstrated between the strength of the NASBA signals and the BI. Furthermore, the usefulness of the detection of 16S rRNA by NASBA to monitor the efficacy of leprosy treatment was investigated using an additional 154 biopsy specimens analyzed from 80 MB patients during the course of treatment. The NASBA positivity rate declined during treatment. A significant decrease was observed after only 1-3 months. These results favor the view that detection of RNA by NASBA may reflect the viability of M. leprae.

Biopsy↗

PubMed1972

Microbial uptake of lead.

Micrococcus luteus and Azotobacter sp. cells grown in broth in contact with a dialysis membrane containing lead bromide were found to immobilize 4.9 and 3.1 x 10(2) milligrams of lead per gram of whole cells, on a dry weight basis, respectively. Culture turbidity and cell count measurements on these and other cell cultures show that lead bromide, lead iodide, and lead bromochloride in concentrations approaching solubility limits have no detectable effect on overall growth rate and cell viability. Analyses of cellular subfractions reveal that fractions of cell wall plus membrane contain 99.3 and 99.1 percent of the lead found associated with Micrococcus luteus and Azotobacter sp., respectively. The remainder is found associated with the cytoplasmic fractions.

Azotobacter↗

PubMed1999

Clostridium difficile toxins may augment bacterial penetration of intestinal epithelium.

BACKGROUND: Clostridium difficile can be recovered from many high-risk hospitalized patients receiving broad-spectrum antibiotic therapy. Clostridium difficile toxins A and B have been associated with increased intestinal permeability in vitro and there is growing evidence that increased intestinal permeability may be a common mechanism whereby enteric bacteria penetrate the intestinal epithelium. HYPOTHESIS: Clostridium difficile-induced alterations in the intestinal barrier facilitate microbial penetration of the intestinal epithelium, which in turn facilitates the translocation of intestinal bacteria. DESIGN: Mature Caco-2 enterocytes were pretreated with varying concentrations of toxin A or toxin B followed by 1 hour of incubation with pure cultures of either Salmonella typhimurium, Escherichia coli, or Proteus mirabilis. The effects of toxins A and B on enterocyte viability, cytoskeletal actin, and ultrastructural topography were assessed using vital dyes, fluorescein-labeled phalloidin, and scanning electron microscopy, respectively. The toxins' effects on bacterial adherence and bacterial internalization by cultured enterocytes were assessed using enzyme-linked immunosorbent assay and quantitative culture, respectively. Epithelial permeability was assessed by changes in transepithelial electrical resistance and by quantifying paracellular bacterial movement through Caco-2 enterocytes cultivated on permeable supports. RESULTS: Neither toxin A nor toxin B had a measurable effect on the numbers of enteric bacteria internalized by Caco-2 enterocytes; however, both toxins were associated with alterations in enterocyte actin, decreased transepithelial electrical resistance, and increased bacterial adherence and paracellular transmigration. CONCLUSION: Clostridium difficile toxins A or B may facilitate bacterial adherence and penetration of the intestinal epithelial barrier.

Bacterial Adhesion↗

PubMed1986

Susceptibility of poultry biting lice (Mallophaga) to Dipel and Bacilan (Bacillus thuringiensis).

Dipel (Abbott Laboratories, N. Chicago, U.S.A.) and Bacilan (PPR, Wałcz, Poland), commercial preparation of Bacillus thuringiensis var. kurstaki, containing 16,000 IU/mg were tested in vitro on Menopon gallinae (L.) and Eomenacanthus stramineus (Nitzsch). The insects were placed in feathers, in Petri plates, and sprayed with 0.25, 0.5, 0.75, 1, 2, 4 and 10% solutions of insecticides. Each concentration was tested on a group of 10 insects, in three replications. The viability was observed after 10, 20, 30, 60 min and then 10 times within 1 hr intervals. Dipel was effective in lower concentrations [LC50 = 2.2 (1.4-3.5)% against E. stramineus and 2.9 (1.8-4.6) against M. gallinae]. LC50 of Bacilan were 2.9 (1.8-4.3) for E. stramineus and 3.2 (2.5-4.0)% for M. gallinae respectively. The ingestion of microbial insecticides was proved by the presence of spores B. thuringiensis in the insects being previously exposed to indirect activity of 2.2% Dipel and 2.9% Bacilan. Moreover, the growth of B. thuringiensis colonies in the lice culture was observed. 3% Dipel solution proved to be effective in killing the insects on the host, but no earlier than after the second application within 24 hr intervals.

Animals↗

PubMed1995

Evaluation of in vitro and in vivo antimicrobial activity of a new topical antiinfective agent.

ST 1103 (Undecyl [4-N,N,N-trimethylammonium-(R)-3- isovaleroyloxy]-butanoate methanesulfonate) is a novel compound endowed with a broad antimicrobial spectrum. ST 1103 is able to inhibit the in vitro growth of Gram-positive bacteria (mean MIC value of 2.60 micrograms/ml), Gram-negative bacteria (mean MIC value of 27.00 micrograms/ml), yeasts and yeast-like fungi (mean MIC value of 3.76 micrograms/ml), filamentous and dermatophytic fungi (mean MIC value of 18.33 micrograms/ml). Since indirect evidence indicates a poor oral absorbtion, ST 1103 was topically administered to mice with skin infections caused by mixed inocula. In these conditions, ST 1103 was able to cure mice infected with T. quinckeanum, S. aureus as well as immunodepressed mice infected with T. quinckeanum, S. aureus and C. albicans. Conversely, miconazole (reference compound) appeared inadequate, in our experimental conditions, for a definitive therapy of the skin mycosis superinfected by staphylococcus. By using an in vitro 3D-human skin model, ST 1103 was fairly well tolerated in terms of both cell viability and release of inflammatory mediators. In a dermal tolerance study in mice, ST 1103 at a concentration of 1% did not show any sign of local irritation on both intact and abraded skin after an 8-day topical treatment. In conclusion, ST 1103 appears to be a promising candidate for treatment of cutaneous infections caused by mixed microbial pathogens.

Administration, Topical↗

PubMed1989

Transient exposure to a physiologically-relevant concentration of calcium confers tobramycin resistance upon sessile cells of Pseudomonas aeruginosa.

Sessile populations of a mucoid Pseudomonas aeruginosa isolate generated in M-56 medium of 'low' (0.02 mM) or 'high' (2.5 mM) Ca2+ were treated with tobramycin (250 micrograms.ml-1), as were biofilms exposed transiently to 'high' Ca2+ medium. Viability decreased by over 99.9% within 8 h in the 'low' and 'high' samples, while transient Ca2+ exposure was protective. Dispersed sessile bacteria were as tobramycin sensitive as planktonic bacteria.

Calcium↗

PubMed1998

Cytotoxicity and mutagenicity of a 2,4,6-trinitrotoluene (TNT) and hexogen contaminated soil in S. typhimurium and mammalian cells.

The toxicity and mutagenicity of aqueous and organic extracts of soil contaminated with TNT, TNT metabolites and hexogen was determined in mammalian cell lines and in prokaryotic cells. The prokaryotic toxicity was determined via the colony forming ability of Salmonella typhimurium (strains TA 98 and TA 100). The same strains were used to test mutagenicity in the Ames test. The mammalian toxicity was analyzed in human fibroblasts by the inhibition of cell growth and cell viability (MTT assay). The mammalian mutagenicity was tested with the HPRT test in V79 cells (hamster lung). The aqueous soil extract did not reveal toxicity or mutagenicity in any of the tests performed. The DMSO/ethanol extract showed toxicity and mutagenicity in S. typhimurium. Thereby strain TA 98 was more sensitive than strain TA 100. In human fibroblasts cell growth was strongly inhibited, whereas no reduction of cell viability was found in the MTT test. Mutagenicity of the DMSO/ethanol extract of the soil was demonstrated in V79 cells.

Animals↗

PubMed2003

Evaluation of the effect of malt, wheat and barley extracts on the viability of potentially probiotic lactic acid bacteria under acidic conditions.

In this work, the effect of cereal extracts, used as delivery vehicles for potentially probiotic lactic acid bacteria (LAB), on the acid tolerance of the cells was evaluated under conditions that simulate the gastric tract. More specifically, the effect of malt, barley and wheat extracts on the viability of Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus reuteri during exposure for 4 h in a phosphate buffer acidified at pH 2.5 was investigated. In the absence of cereal extracts all strains demonstrated a significant reduction in their cell population, particularly L. plantarum. The viability of L. plantarum was improved by approximately 4 log(10) cycles in the presence of malt and 3 log(10) cycles in the presence of wheat and barley. The survival of L. acidophilus and L. reuteri was increased by more than 1.5 and 0.7 log(10) cycle, respectively, upon addition of cereal extracts. In order to evaluate the contribution of the cereal constituents on cell survival, the individual effect of glucose, maltose and free amino nitrogen (FAN), which were added at concentrations that correlated to the reducing sugar and FAN content of the cereal extracts, was examined. The viability of L. plantarum was progressively improved as the maltose or glucose concentration increased; an increase by approximately 2 log(10) cycles was observed in the presence of 8.33 g/l sugar. The survival of L. acidophilus increased by more than 1 log(10) cycle, even at very low concentrations of maltose and glucose (e.g., 0.67 g/l), while L. reuteri stability was enhanced in the presence of maltose but no appreciable effect was demonstrated in the presence of glucose. Sugar analysis indicated that glycolysis was inhibited in all cases. Addition of tryptone and yeast extract, used as sources of FAN, enhanced L. acidophilus acid tolerance, but did not affect L. reuteri and L. plantarum. The results presented in this study indicate that malt, wheat and barley extracts exhibit a significant protective effect on the viability of L. plantarum, L. acidophilus and L. reuteri under acidic conditions, which could be mainly attributed to the amount of sugar present in the cereal extracts.

Analysis of Variance↗

PubMed1997

Activity of quinupristin/dalfopristin against Streptococcus pneumoniae in vitro and in vivo in the rabbit model of experimental meningitis.

We tested the efficacy of quinupristin/dalfopristin, an antibiotic made up of dalfopristin (70%) and quinupristin (30%) against a large panel of Streptococcus pneumoniae strains. The pneumococcal isolates (217) included 200 penicillin-resistant and 17 penicillin-susceptible clinical isolates. Eighty-nine of the 200 resistant bacteria showed an intermediate level and 111/200 showed a high level of resistance to penicillin. Of the highly resistant strains, 56/111 belonged to the multidrug-resistant Spanish/USA epidemic clone of S. pneumoniae, as defined by appropriate genetic techniques. The resistant panel also included six isolates of another multidrug-resistant epidemic clone: isolates with capsular type 6B belonging to the Spanish/Icelandic clone of S. pneumoniae. Quinupristin/dalfopristin had a uniform mean MIC of 0.25 mg/L against all pneumococcal isolates, including 37 strains representing a wide spectrum of erythromycin MICs, from 0.03 up to 8.0 mg/L. Quinupristin/dalfopristin showed powerful bactericidal activity against a penicillin-susceptible test strain in vitro and against representatives of both the Spanish/USA and the Spanish/Icelandic multidrug-resistant clones. The rate of bactericidal activity was independent of drug concentration between 2.5 x and 10 x MIC. Quinupristin/dalfopristin was also tested in a rabbit model of experimental meningitis using 50 mg/kg i.v. bolus injections and a penicillin-susceptible capsular type 3 S. pneumoniae strain as the test organism. Quinupristin/dalfopristin had no effect on the intracisternal growth of bacteria when the drug was injected before CSF inflammation, whereas it caused a 2 log kill in 2 h, after which bacterial growth in the CSF resumed, when injected i.v. at a time of inflammation. When a second dose was given 2 h later, this produced a 3 log loss of viability after 4 h. A single injection of ampicillin 50 mg/kg i.v. caused a similar 3 log kill after 4 h under comparable conditions.

Animals↗

PubMed1989

Inhibition by antibiotics of the bacterial response to long-term starvation of Salmonella typhimurium and the colon microbiota of mice.

The number of viable cells of two strains of Salmonella typhimurium and the number of viable cells and the cell size of the colon microbiota of mice were examined during non-growing conditions after exposure to antibiotics with known modes of action. Salmonella typhimurium starved for 1, 2, 4, 5, 12 and 20 d in a phosphate buffer saline solution and subsequently exposed for 2 and 6 h showed the following characteristics. The protein synthesis inhibitors gentamicin and tetracycline, the RNA synthesis inhibitor rifampicin and the membrane potential inhibitor polymyxin all impaired survival of starved cells. The reduction in the number of viable cells caused by the addition of gentamicin, rifampicin and polymyxin was generally more pronounced with extended exposure to energy and nutrient deprivation. Both 2- and 6-h exposure of tetracycline, however, had diminishing inhibitory effects after 20 d compared with 5 d of starvation. Control experiments to verify non-growing conditions in the starvation regime showed that DNA and cell wall synthesis inhibitors had no inhibitory effect after 24-h starvation. The rough mutant strain displayed a lower sensitivity to a hydrophobic rather than a hydrophilic inhibitor as compared to the smooth wild-type strain. The cell size reduction but not viability was partly prevented by protein synthesis inhibitors as seen for both in vivo and in vitro colon microbiota studies.

Animals↗

PubMed1989

Evaluation of two multidrug regimen in hospitalised multibacillary cases.

Fifty three multibacillary leprosy cases were treated with two regimens of MDT L1 consisting of Rifampicin, Dapsone and Ethionamide and L2 consisting of Rifampicin, Dapsone and clofazimine. The results were compared at regular intervals and at the end of the study (24 months). Clinical inactivity, bacteriological negativity, ENL reactions, upgrading reactions were seen in L1 group in 65%, 4.54%, 50% and 41% of cases respectively while 65%, 25.8%, 30% and 45% respectively in L2 regimen group. Zero percent morphological Index was achieved in all cases in L1 regimen 90% in L2 regimen cases. No viability was found on mouse foot pad inoculation after 6 months in L1 while after 18 months in L2 cases.

Clofazimine↗

PubMed1996

Why do many ruminal bacteria die and lyse so quickly?

Studies using 15N have indicated that as much as 50% of the microbial mass turns over before N passes to the lower gut, and this N recycling significantly decreases the availability of microbial protein. Protozoa digest bacteria and smaller protozoa, but bacterial protein can turn over even if protozoa are not present. Fibrobacter succinogenes cultures lyse even when they are growing, and the lysis rate is independent of growth rate. When extracellular sugar is depleted, F. succinogenes secretes an extracellular proteinase that inactivates the autolysins. This method of autolytic regulation decreases the turnover of stationary cells. Bacteriophage and anaeroplasma can cause lysogeny, but, as yet, there is little proof that these processes are important determinants of bacterial turnover in vivo. Dietary manipulations (e.g., salt feeding and particle size reduction) that increase liquid and solid dilution rates can increase bacterial flow by decreasing bacterial residence time and turnover. Some dead ruminal bacteria are able to maintain their cellular integrity, and the ratio of dead to live cells in ruminal fluid may be as great as 10:1. Bacterial survival appears to be at least partially explained by the method of sugar transport. When bacteria rely solely on mechanisms of ion-coupled sugar symport, an energized membrane is necessary for the reinitiation of growth. If group translocation (phosphotransferase system) is the mechanisms of transport, uptake can be driven by phosphoenolpyruvate, and an energized membrane and the storage of intracellular reserve materials are not an absolute criteria for survival. In some cases, N deprivation accelerates death. When Prevotella ruminicola was limited for N under conditions of excess energy, methylglyoxal production caused a rapid decrease in viability. The impact of bacterial death in the rumen is not clear-cut. If the rate of fermentation is zero-order with respect to cell concentration (substrate-limited), cell death would have little impact on digestion.

Animals↗

PubMed1979

In vitro survival of human pathogenic fungi in Hawaiian beach sand.

In vitro studies utilizing 4 pathogenic fungi, Trichosporon cutaneum, Candida albicans, Microsporum gypseum and Trichophyton mentagrophytes, all known from Hawaiian beaches, indicate that they survive in the fluctuating beach habitat where they can serve as potential sources of infection for significant periods of time. Survival was measured by testing the viability of propagules at intervals for 6 months. All species survived 6 months under 1 or more experimental conditions. Survival patterns showed both increases and decreases depending upon the given parameters designed to simulate various beach conditions. Propagules inoculated on hair and horn (keratinized inoculum) did not remain viable longer than propagules from pure culture suspensions (non-keratinized). Microbial antagonism was not a major factor in survival. All species survived at least 1 month in non-sterile sand inoculated with keratinized propagules. This condition approximated the natural sand habitat. Alternate wetting and drying of sand caused an overall decrease in survival time except for M. gypseum (non-keratinized inoculum) at 37 degrees C in sterile sand and T. mentagrophytes (keratinized inoculum) at 37 degrees C in non-sterile sand. Temperature was important: increasing temperature resulted in a general decrease in survival time; 45 degrees C was definitely inhibitory, with the exception of T. cutaneum which survived that level for 6 months (keratinized inoculum). Salinity did not influence survival.

Candida albicans↗

PubMed1999

Vaccine protocols to optimise the protective efficacy of BCG.

SETTING: A deer model has been developed to study protection produced with BCG vaccination, against infection and the development of pathology, following experimental intratonsilar infection with virulent Mycobacterium bovis. OBJECTIVE: To determine how the dose of vaccine, the route of vaccination, the viability of the vaccine and exposure to glucocorticoids at the time of vaccination, may affect the protective efficacy of BCG vaccines. DESIGN: Deer were vaccinated with BCG and later challenged with virulent M. bovis via the tonsilar route. Protection against infection and development of disease was evaluated at necropsy six months after challenge with M. bovis, by histological examination and microbial culture. RESULTS: Significant protection against infection and disease were obtained following boosting with two low doses (5 x 10(4) cfu) or moderate doses (5 x 10(7) cfu) of live (freshly cultured and lyophilized) BCG. Inferior levels of protection were obtained with high dose (5 x 10(8) cfu) of live BCG. Similar levels of protection were found with vaccines given subcutaneously or via the tonsilar route. Killed vaccine in a mineral-oil adjuvant did not evoke protective immunity and treatment with dexamethasone prior to vaccination with live BCG ablated its efficacy. Protection against infection did not correlate with skin test delayed type hypersensitivity (DTH) or lymphocyte transformation to tuberculin. CONCLUSIONS: Two doses of live BCG gave significant protection against experimental infection and disease caused by virulent M. bovis. Single dose vaccine protected against disease but not infection. Vaccines administered at a dosage which did not evoke DTH, provided protection against tuberculosis infection and disease.

Adjuvants, Immunologic↗

PubMed2003

Assessment of the antibacterial activity of selected flavonoids and consideration of discrepancies between previous reports.

Activity of the flavonoids apigenin, baicalin and galangin against sensitive and antibiotic resistant strains of Staphylococculs aureus, Enterococcus faecalis, E. faecium, Escherichia coli and Pseudomonas aeruginosa was investigated. Using an agar dilution assay, galangin was shown to have a minimum inhibitory concentration (MIC) of 25 to 50 microg/mL against all six strains of S. aureus but negligible activity against the othe species. Apigenin displayed only marginal activity against S. aureus and no activity was detected from baicalin. In inhibition curve studies, galangin caused a 100,000-fold decrease in the viability of a growing population of S. aureus NCTC 6571 within the first two hours of treatment. Decreases in viability of S. aureus NCTC 11561 and NCIMB 9968 populations were also observed.

Anti-Infective Agents↗

PubMed2004

Comparison of application methods to prolong the survival of potential biocontrol bacteria on stored sugar-beet seed.

AIMS: To develop bacterial inoculation treatments on sugar-beet seed that will maintain a commercially acceptable degree of viability for a minimum of 4 months storage at ambient temperature. METHODS AND RESULTS: Single rifampicin-resistant (Rif(+)) strains of both Gram-positive and negative bacterial isolates (mostly pseudomonads) were applied in turn to sugar-beet seed in a comparative study by seed soaking, encapsulation in alginate, pelleting using an inoculated peat carrier or seed priming. The treated seed was assessed for bacterial survival over a time course by plating out homogenized samples onto a selective medium. Priming inoculation offered a significant improvement over all the other application strategies tested. After pelleting with fungicides and drying at 40 degrees C, Pseudomonas marginalis/putida P1W1 maintained populations of >6.6 log(10) CFU g(-1) seed during 4 months storage at 15 degrees C. Subsequent experiments verified a stabilized population under these storage conditions with commercial pellets at <7% moisture content. CONCLUSION: An inoculation method was established which allowed the survival on seed of a Gram-negative bacterium at ambient temperature with little loss in viability. SIGNIFICANCE AND IMPACT OF THE STUDY: This has promising implications for the delivery of beneficial bacteria, especially Gram-negative strains, on sugar beet.

Bacterial Physiological Phenomena↗

PubMed2003

Detection and fate of Bacillus anthracis (Sterne) vegetative cells and spores added to bulk tank milk.

A preparation of Bacillus anthracis (Sterne strain) spores was used to evaluate commercially available reagents and portable equipment for detecting anthrax contamination by using real-time PCR and was used to assess the fate of spores added directly to bulk tank milk. The Ruggedized Advanced Pathogen Identification Device (RAPID) was employed to detect spores in raw milk down to a concentration of 2,500 spores per ml. Commercially available primers and probes developed to detect either the protective antigen gene or the lethal factor gene both provided easily read positive signals with the RAPID following extraction from milk with a commercially available DNA extraction kit. Nucleotide sequence analysis of the vrrA gene with the use of DNA extracted from spiked milk provided molecular data that readily identified the spores as B. anthracis with a 100% BLAST match to the Sterne and Ames strains and easily distinguished them from B. cereus. Physical-fate and thermal-stability studies demonstrated that spores and vegetative cells have a strong affinity for the cream fraction of whole milk. A single treatment at standard pasteurization temperatures, while 100% lethal to vegetative cells, had no effect on spore viability even 14 days after the treatment. Twenty-four hours after the first treatment, a second treatment at 72 degrees C for 15 s reduced the viability of the population by ca. 99% but still did not kill all of the spores. From these studies, we conclude that standard pasteurization techniques for milk would have little effect on the viability of B. anthracis spores and that raw or pasteurized milk poses no obstacles to the rapid detection of the spores by molecular techniques.

Animals↗